WO2021226117A1 - Procédé de transformation d'organismes photosynthétiques - Google Patents

Procédé de transformation d'organismes photosynthétiques Download PDF

Info

Publication number
WO2021226117A1
WO2021226117A1 PCT/US2021/030693 US2021030693W WO2021226117A1 WO 2021226117 A1 WO2021226117 A1 WO 2021226117A1 US 2021030693 W US2021030693 W US 2021030693W WO 2021226117 A1 WO2021226117 A1 WO 2021226117A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
methylation
dam
transforming
dna fragment
Prior art date
Application number
PCT/US2021/030693
Other languages
English (en)
Inventor
John H. Verruto
Jessica N. WEIR
Original Assignee
Synthetic Genomics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Synthetic Genomics, Inc. filed Critical Synthetic Genomics, Inc.
Publication of WO2021226117A1 publication Critical patent/WO2021226117A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01072—Site-specific DNA-methyltransferase (adenine-specific) (2.1.1.72)

Definitions

  • the invention involves the transformation of photosynthetic organisms with DNA molecules.
  • Eukaryotic algae are a diverse group of organisms of great ecological and commercial importance. Transformation of these organisms is complicated by the presence of a cell wall and has been accompanied by problems with efficiency, integration, or stability of transgenes. As a result, genetic transformation of algal species has lagged behind that of other organisms. Nevertheless, some techniques do exist for certain algae, but a lack of knowledge about the requirements for transforming algae has resulted in poor efficiency and other disadvantages. For transformations into many algae PCR products have been found to provide very poor rates of transformation and colony formation.
  • the invention provides methods of transforming photosynthetic organisms, such as green algae.
  • the methods involve contacting one or more DNA fragments that comprise a DNA construct with a methylating enzyme to thereby methylate the one or more DNA fragments, and transforming the one or more DNA fragments into the photosynthetic organism.
  • the DNA fragments that comprise the construct can be the product of a DNA amplification procedure (e.g. PCR or a variant of PCR).
  • the one or more fragments of DNA that comprise a DNA construct are dam methylated prior to being transformed into the photosynthetic organism.
  • the invention provides methods of transforming a photosynthetic organism.
  • the methods involve contacting at least one DNA fragment with a DNA methylating enzyme to produce at least one methylated DNA fragment; and transforming the photosynthetic organism with the at least one methylated DNA fragment.
  • the contacting can be done in vitro.
  • the DNA methylating enzyme can be a methyltranferase.
  • the DNA methylating enzyme can perform m6A methylation.
  • the DNA methylating enzyme can be a prokaryotic DNA methylating enzyme.
  • the methylating enzyme can be a methyltransferase performs dam methylation.
  • the at least one DNA fragment is a plurality of DNA fragments that together comprise a DNA construct.
  • the at least one DNA fragment can be assembled into the DNA construct prior to transforming the photosynthetic organism, or can be transformed as DNA fragments and assembled in the host cell or organism.
  • the at least one DNA fragment can be the product of digestion with a restriction endonuclease.
  • the transforming can be performed using a biolistic method.
  • the photosynthetic organism can be a Chlorophyte alga.
  • the Chlorophyte alga can be an alga of the Class Trebouxiophyceae.
  • the alga of the Class Trebouxiophyceae alga is of the family Oocystacea.
  • the alga is of the genus Oocystis.
  • transforming the at least one methylated DNA fragment is performed by particle bombardment.
  • the transformation construct can be a plasmid having any one or more of a promoter, a gene of interest, and a terminator.
  • the transformation construct can also have an origin of replication and/or a selectable marker.
  • the transformation construct can also have all of a promoter, a gene of interest, a terminator, an origin of replication, and a selectable marker, or any possible combination or sub combination of them.
  • the at least one DNA fragment is provided as a linear fragment.
  • the at least one DNA fragment can be dam methylated.
  • the at least one DNA fragment is not CpG methylated.
  • the methylase can be a deoxyadenosine (Dam) methylase.
  • Figure 1 provides an illustration of an uncut pUK-derived vector containing the integration fragment.
  • Figure 2 provides a schematic illustration of a linearized fragment of the plasmid from Figure 1 that was transformed into Oocystis sp. and showing possible methylation sites.
  • Figure 3 provides a schematic illustration of possible methylation sites for dam, dcm, and CpG methyltransferases on the integration fragment transformed into Oocystis sp. dcml (CCAGG), dcm2 (CCTGG), and CpG (CG) sites are indicated.
  • Figure 4 is an illustration of restriction digests confirming complete methylation of DNA fragments.
  • the top three gels are plasmid constructs that are a) Pad digested gel-purified, b) Pad digested, gel-purified, CpG+, and c) Pad digested gel-purified, dam negative-dcm negative.
  • the bottom four gels are PCR amplified constructs: d) PCR-amplified fragments, e) PCR amplified, dam+, f) PCR amplified CpG+, and g) PCR-amplified CpG+, dam+.
  • the lanes are 1. Uncut, 2. Dpnl, 3. DpnII, 4. Hpall, and 5. Mspl, in that order.
  • the invention discloses methods of transforming photosynthetic organisms.
  • the methods involve the methylation of DNA constructs or parts of DNA constructs, which in some embodiments can be done in vitro, before transformation into photosynthetic microorganisms.
  • the invention provides high rates of successful transformation and colony formation.
  • the present inventors discovered unexpectedly that when a DNA construct (or the parts or fragments of a DNA construct) is subjected to methylation prior to being transformed into photosynthetic microorganisms, much higher rates of transformation and colony formation (containing the construct) were observed.
  • This discovery enables the use of PCR products in the transformation of photosynthetic and green algae.
  • the invention therefore allows for the fast generation of specifically tailored DNA by PCR and its subsequent transformation into photosynthetic microorganisms with high transformation and colony formation rates.
  • the need for time-consuming cloning processes to generate DNA constructs for each target of homologous recombination can therefore be eliminated.
  • DNA methylation is a process by which methyl groups are added to a DNA molecule. Such methylation can change the activity of DNA.
  • DNA methylation in bacteria is important in gene expression and DNA replication.
  • DNA methylation has been believed to be important in prokaryotic organisms. When occurring in eukaryotes, DNA methylation has been believed to occur primarily or exclusively at the cytosine residue of CpG sequences, leading to the formation of 5-methylcytosine.
  • Prokaryotes e.g. E. coli
  • DNA methyltransferases can transfer a methyl group from S-adenosylmethionine to either adenine or cytosine residues.
  • CpG methyltransferases e.g. Dnmtl
  • CG cytosine residues
  • Methylating enzymes include methylases and methyltransferases, both of which can find use in the invention. There are many methylating enzymes and those disclosed here are provided only as examples.
  • the methylating enzyme methylates the N6 position of adenine in GATC sequences; the methylating enzyme can be encoded by the dam gene.
  • a methylating enzyme that methylates 6-adenine e.g. in the sequence GATC
  • m6A methylase i.e. performs m6A methylation
  • the methylating enzyme is an m6A methylase.
  • the at least one DNA fragment can be methylated by contact with a dam methylase.
  • the at least one DNA fragment can be dam methylated.
  • the DNA methylating enzyme can be a prokaryotic DNA methylating enzyme, such as any described herein.
  • a prokaryotic DNA methylating enzyme is one naturally found in prokaryotic organisms.
  • the DNA methylating enzyme can be a heterologous DNA methylating enzyme.
  • the at least one DNA fragment can lack CpG methylation. Any of the methylating enzymes disclosed herein can be utilized in the invention.
  • the at least one DNA fragment contacted with the DNA methylating enzyme is a product of PCR or a PCR variant, non-limiting examples of which include multiplex PCR, asymmetric PCR, nested PCR, quantitative PCR, hot-start PCR, touchdown PCR, assembly PCR, colony PCR, the digital polymerase reaction, suicide PCR, COLD-PCR, or another amplification procedure.
  • the DNA fragment can be unmethylated, or not have been subjected to a methylation procedure.
  • the DNA fragment contacted with the DNA methylating enzyme is an unmethylated fragment.
  • the invention provides methods of transforming photosynthetic organisms.
  • the photosynthetic microorganism can be a microalga or a green alga.
  • the photosynthetic organism can be a recombinant microorganism.
  • the photosynthetic microorganism can be any eukaryotic alga or microalga such as, but not limited to, a Chlorophyte, an Ochrophyte, or a Charophyte alga.
  • the microorganism can be a Chlorophyte alga of the taxonomic Class Chlorophyceace, or of the Class Chlorodendrophyceae, or the Class Prasinophyceace, or the Class Trebouxiophyceae, or the Class Eustigmatophyceae.
  • the microorganism can be a member of the Class Chlorophyceace, such as a species of any one or more of the genera Asteromonas, Ankistrodesmus, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorodendrales, Chloroellales, Chrysosphaera, Dunaliella, Haematococcus,
  • Class Chlorophyceace such as a species of any one or more of the genera Asteromonas, Ankistrodesmus, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorodendrales, Chloroellales, Chrysosphaera, Dunaliella, Haematococcus,
  • the microalga can be a member of the Class Chlorodendrophyceae, such as a species of any one or more of the genera Prasinocladus , Scherffelia , or Tetraselmis.
  • the alga can be a member of the Class Prasinophyceace, optionally a species of any one or more of the genera Ostreococcus or Micromonas.
  • the microalga can be a member of the Class Trebouxiophyceae, and optionally of the Order Chlorellales, and optionally a genera selected from any one or more of Botryococcus, Chlorella, Auxenochlorella, Heveochlorella, Marinichlorella, Oocystis, Parachlorella, Pseudochlorella, Tetrachlorella, Eremosphaera, Franceia, Micractinium, Nannochloris, Picochlorum, Prototheca, Stichococcus, or Viridiella , or any of all possible combinations or sub-combination of the genera.
  • the alga is a Chlorophyte alga of any of Class Trebouxiophyceae, the Order Chlorellales, the Family Oocystaceae, Chlorellaceae, or Eustigmatophyceae, and optionally a genera selected from one or more of Oocystis, Parachlorella, Picochlorum, Nannochloropsis , and Tetraselmis in all possible combinations or sub-combinations.
  • the alga can also be from the genus Oocystis , or the genus Parachlorella , or the genus Picochlorum , or the genus Tetraselmis , or from any of all possible combinations and sub-combinations of these genera.
  • DNA constructs are molecules of DNA constructed using molecular biology techniques that can be transformed into a targeted microorganism.
  • the DNA construct is a plasmid.
  • a DNA construct can be provided as one or more DNA fragments.
  • the plasmid can be useful for transforming algal cells.
  • DNA constructs can have one or more of a promoter sequence, a gene of interest (GOI), and a transcription terminator or polyadenylation signal sequence (poly-A), or all of these structures.
  • DNA constructs can also, optionally, have a selectable marker (e.g. one or more antibiotic resistance genes) and/or an origin of replication.
  • the DNA construct can comprise a single DNA fragment or can be comprised of a plurality of DNA fragments.
  • Each DNA fragment in a plurality of DNA fragments can comprise a portion of a DNA construct.
  • the plurality of DNA fragments can be assembled prior to transformation into a cell or microorganism.
  • the assembly of DNA constructs from a plurality of DNA fragments is required in many applications of molecular biology, and many such techniques are known.
  • the DNA construct can also be assembled from the fragments by the cell or microorganism.
  • the DNA construct can be assembled from 4 or more, or 6 or more, or 8 or more fragments or 10 or less, or 20 or less or 25 or less or 30 or less or 2-10 or 2-20 or 2-30 or more than 30 fragments of DNA.
  • a transformation construct is a vehicle for carrying genetic material into a cell.
  • the genetic material can be heterologous DNA.
  • Heterologous DNA is a sequence of DNA from a species or origin other than the cell or organism the DNA is introduced into. Heterologous sequences can also be synthetic and not derived from the cell or organism.
  • a transformation construct can be a linear fragment or a circular DNA. In any embodiment the DNA construct can be transformed into the cell or microorganism as one or more linear DNA fragments or as circular DNA.
  • the DNA fragments that are transformed into the photosynthetic organism of the invention are methylated.
  • the methylation can be done enzymatically and in vitro, or by any method described herein.
  • the DNA fragments are products of a DNA amplification procedure, for example PCR or a variant of PCR.
  • the fragments can be methylated as disclosed herein after performing a DNA amplification procedure.
  • the DNA fragments transformed into the photosynthetic organism of the invention can be the products of cloning in an organism that does not methylate DNA.
  • the DNA fragments transformed into the photosynthetic organism of the invention can be the products of cloning in an organism that does not dam methylate DNA.
  • the DNA fragments transformed into the photosynthetic organism of the invention can be the products of cloning in an organism that does not have a dam methylase.
  • the DNA fragments can be the product of digestion with one or more restriction endonuclease(s).
  • the DNA fragments can be methylated by an enzyme that is not endogenous to the organism the DNA is transformed into in a later step, or that is not endogenous to an organism the DNA was amplified in.
  • Transformation refers to the process of introducing exogenous DNA into cells, which can be plant or algal cells. Transformation of a cell or microorganism of the invention can occur by any convenient means. In various embodiments electroporation or particle bombardment methods can be used to accomplish transformation. In any embodiment the cells transformed in the method can be competent cells. In some embodiments the DNA fragments comprising the DNA construct can be methylated prior to transformation. Persons of ordinary skill with resort to this disclosure will realize that cells can be made competent according to various methods, for example by electroporation or heat shock of chemically prepared competent cells; for example cells can be grown up in culture and harvested in mid log phase.
  • harvested cells can be washed with cold water or sorbitol by repeatedly pelleting and resuspension to remove salts and other components that could interfere with transformation, with final pelleting and resuspension in glycerol.
  • cells When cells are to be transformed by heat shock they can be incubated in calcium chloride, which can be supplemented with other ions (e.g. manganese, potassium, cobalt, rubidium, dimethylsulfoxide and dithiothreitol). Other methods of preparing competent cells can also be used.
  • Methods of transformation can include any suitable method.
  • transformation can be accomplished by biolistic methods, electroporation, or heat shock of chemically prepared competent cells.
  • Electroporation can involve the use of a commercially available electroporator to expose cells to a brief pulse of a high-voltage electric field resulting in a temporary rearrangement of the cell membrane and membrane permeability.
  • the electric pulse can utilize exponential decay (or capacitance discharge), where a set voltage is applied and allowed to decay over a few milliseconds.
  • heat shock method can be used to transform the cells.
  • heat shock can be performed at 37-42 °C for 25-45 seconds, but persons of ordinary skill in the art with resort to the present disclosure will realize other effective protocols.
  • Methylation can be performed by contacting at least one DNA fragment described herein with a DNA methylating enzyme described herein to produce at least one methylated DNA fragment.
  • the methylation can be performed using any method described herein.
  • the contacting can be done in vitro.
  • the at least one methylated DNA fragment is thereby transformed into the cell or organism of the invention.
  • a pUC19-based vector was constructed containing a selectable marker cassette encoding a blasticidin resistance gene flanked by 500 bp of Oocystis genomic sequence on either side and controlled by endogenous promoters and terminators, all within restriction sites for Pad restriction enzyme.
  • This vector was initially constructed in E. coli DH5a cells, which perform both dam and dcm methylation (Me positive).
  • the base vector was also purified using a commercially available “miniprep” kit and transformed into competent E. coli cells negative for both dam and dcm methylation enzymes (dam-, dcm-) using standard methods (Me negative) provided by the manufacturer.
  • Methylation treatments were done using enzymes sourced from commercial sources and according to manufacturer suggested protocols. Dam methylation was performed with dam methyltransferase sourced from E. coli carrying the plasmid pTP166, which had the dam modification gene. CpG methylation was performed by the CpG methyltransferase (M.SssI). Methylation was tested for completion by restriction digestion with Dpnl, DpnII, Hpall and MspT Dpnl and DpnII have the same recognition sequence (GATC) but Dpnl only cuts dam-methylated DNA (GATC) and is blocked by overlapping CpG methylation, while DpnII is blocked by dam methylation.
  • GTC recognition sequence
  • Hpall and Mspl have the same recognition sequence (CCGG) but Hpall is blocked by CpG methylation and Mspl is not blocked by any methylation.
  • M.SssI is a CpG methyltransferase.
  • Figure 4 shows seven gels, which are
  • DNA fragments were transformed into Oocystis sp. using a gene gun. DNA was precipitated onto gold particles, which were adhered to the inside of tubing, and helium gas was fired through the tubing to project the DNA-coated gold particles into cells adhered on solid non-selective media. The following day the cells were moved onto selective media for growth and screening of transformed colonies. Colonies were counted after seven days to compare the effects of DNA methylation on transformation efficiency.
  • Table 1 Colony counts from transformation into Oocystis sp.
  • the experiment was expanded to include treatments with additional methylating enzymes that have different methylation patterns.
  • the additional methylating enzymes used were EcoGII, Alul, Haelll, and Mspl.
  • EcoGII adds m6A methylation (providing N6-methyl adenosine) to adenines without sequence specificity; Alul and Haelll add non-CpG methylation (to 22 and 30 possible sites, respectively), and Mspl was also used, which performs CHG methylation.
  • dam negative/dcm negative (dam , dcm ) plasmid was subjected to either a step of dam methylation or to no treatment before being re-transformed into Oocystis sp.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Physiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des procédés de transformation d'organismes photosynthétiques, tels que des algues vertes. Ces procédés consistent à méthyler au moins un fragment d'ADN d'une construction génétique et à transformer l'au moins un fragment en l'organisme photosynthétique. Les fragments d'ADN peuvent être le produit d'une procédure d'amplification d'ADN, telle qu'une PCR ou une procédure de type PCR. Dans un mode de réalisation, l'au moins un fragment d'ADN, qui comprend une construction génétique, est déméthylé par dam avant d'être transformé en organisme photosynthétique.
PCT/US2021/030693 2020-05-05 2021-05-04 Procédé de transformation d'organismes photosynthétiques WO2021226117A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063020414P 2020-05-05 2020-05-05
US63/020,414 2020-05-05

Publications (1)

Publication Number Publication Date
WO2021226117A1 true WO2021226117A1 (fr) 2021-11-11

Family

ID=78412293

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/030693 WO2021226117A1 (fr) 2020-05-05 2021-05-04 Procédé de transformation d'organismes photosynthétiques

Country Status (2)

Country Link
US (1) US20210348183A1 (fr)
WO (1) WO2021226117A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070022485A1 (en) * 2003-07-08 2007-01-25 Japan Science Techonology Agency Method of preparing transgenic organism with use of methylation and system therefor
US20110053273A1 (en) * 2007-10-08 2011-03-03 Synthetic Genomics, Inc. Methods for cloning and manipulating genomes
US20160060643A1 (en) * 2014-09-03 2016-03-03 Arizona Board Of Regents On Behalf Of Arizona State University Premethylation of dna for high efficiency transformation of cyanobacteria

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008522619A (ja) * 2004-12-06 2008-07-03 エモリー ユニヴァーシティ 細菌ｄａｍｄｎａメチルトランスフェラーゼの小分子阻害剤
AU2017211359A1 (en) * 2016-01-27 2018-08-02 Total Raffinage Chimie Multigene expression in microalgae
EP3921416A1 (fr) * 2019-02-06 2021-12-15 Fred Hutchinson Cancer Research Center Bactéries productrices de minicercle conçues pour produire de manière différentielle des molécules d'acide nucléique à l'intérieur de celles-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070022485A1 (en) * 2003-07-08 2007-01-25 Japan Science Techonology Agency Method of preparing transgenic organism with use of methylation and system therefor
US20110053273A1 (en) * 2007-10-08 2011-03-03 Synthetic Genomics, Inc. Methods for cloning and manipulating genomes
US20160060643A1 (en) * 2014-09-03 2016-03-03 Arizona Board Of Regents On Behalf Of Arizona State University Premethylation of dna for high efficiency transformation of cyanobacteria

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BO WANG, YU JIANPING, ZHANG WEIWEN, MELDRUM DEIRDRE R.: "Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 81, no. 24, 15 December 2015 (2015-12-15), US , pages 8500 - 8506, XP055694451, ISSN: 0099-2240, DOI: 10.1128/AEM.02575-15 *
KIM ET AL.: "Improvement of Transformation Efficiency Through In Vitro Methylation and Sacll Site Mutation of Plasmid Vector in Bifidobacterium longum MG 1", JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 20, no. 6, 19 April 2010 (2010-04-19), pages 1022 - 1026, XP055871153 *

Also Published As

Publication number Publication date
US20210348183A1 (en) 2021-11-11

Similar Documents

Publication Publication Date Title
Char et al. An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize
Schiml et al. Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas
Du et al. Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9
Quétier The CRISPR-Cas9 technology: closer to the ultimate toolkit for targeted genome editing
US20200181591A1 (en) Compositions and methods for site-directed dna nicking and cleaving
Svitashev et al. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA
CN105132451B (zh) 一种CRISPR/Cas9单一转录单元定向修饰骨架载体及其应用
CN113789317A (zh) 使用空肠弯曲杆菌crispr/cas系统衍生的rna引导的工程化核酸酶的基因编辑
US20200080110A1 (en) Method for targeted alteration of duplex dna
Hsu et al. Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco
CN110891965A (zh) 植物中使用的抗crispr蛋白的方法和组合物
WO2021226363A1 (fr) Enzymes à domaines ruvc
CN111979264B (zh) 基于CRISPR/Cas9系统对博落回PDS基因编辑体系的构建方法及应用
JP6994730B2 (ja) ゲノム編集タンパク質の直接導入による糸状菌ゲノム編集方法
CN114075559A (zh) 一种2型CRISPR/Cas9基因编辑系统及其应用
Wang et al. Targeted mutagenesis in hexaploid bread wheat using the TALEN and CRISPR/Cas systems
US20210348183A1 (en) Method of transforming photosynthetic organisms
AU2022335499A1 (en) Enzymes with ruvc domains
Bhandawat et al. Biolistic delivery of programmable nuclease (CRISPR/Cas9) in bread wheat
CN104388462A (zh) 一种小麦转化双t超毒载体及应用
Chopy et al. Genome editing by CRISPR-Cas9 technology in Petunia hybrida
JP2022516025A (ja) 遺伝子的に改変されたｃｌｏｓｔｒｉｄｉｕｍ細菌の、調製およびその使用。
Viviani et al. Origin of the genome editing systems: application for crop improvement
Klimek-Chodacka et al. Carrot genome editing using CRISPR-based systems
US20230272408A1 (en) Plastid transformation by complementation of plastid mutations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21799505

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21799505

Country of ref document: EP

Kind code of ref document: A1